Antimicrobial activities of different solvent extracts from stem and seeds of Peganum Harmala L

Wild medicinal herbs have been used as folk and traditional medicines all across the world since well before recorded history. This present study was designed to test the antimicrobial activities of five different solvent extracted samples ( n-hexane , n-butanol , ethyl acetate , methanol , and water ) of Peganum harmala using stems and seeds. Two different strains of Gram-negative bacteria ( Escherichia coli and Klebsiella pneumonia) , two Gram-positive bacteria (Bacillus subtilus and Staphylococcus aureus) , and one fungal strain ( Candida albicans ) were used. The antimicrobial activities were measured using a disc diffusion assay. Two concentrations of the extracts (1 and 2mgDisc -1 ) were used. Ethyl acetate fraction was found more affective among the tested solvents and showed maximum activity (zone of inhibition) against S . aureus ( 65.53 and 81.10% ) , E . coli ( 46.22 and 61.29% ) while n-butanol and water fractions gave maximum activity against S . aureus (78.86 and 70.00%) and K . pneumonia (57.00 and 61.39%) respectively. Water fraction showed maximum activity against C . albicans (60.00 and 81.88%). In the case of the stem, Ethyl acetate again showed more activity against B . subtilus (38.57 and 42.10%) and S. aureus (36.66 and 46.66%) while n-butanol showed maximum activity against K . pneumonia (24.55 and 32.44%) and E . coli (27.93 and 37.61%). Methanol was found more effective against C . albicans (25.71 and 43.80%). Seed extracted samples were found more effective compared to the stem. Ethyl acetate , butanol , and aqueous extracted samples showed good activity against the tested

microbes, so these fractions are recommended for study their mechanism of actions and isolation of bioactive metabolites responsible for antimicrobial activities. The P. harmala should be evaluated for their bioactive compounds to be used in future studies. Our objective is to provide the framework for future study on the roles of P. harmala as traditional medicines.

Introduction
Our environment is teeming with harmful germs, and plants and animals are vulnerable to infection [1]. Attempts are being undertaken in a variety of ways to prevent and control these diseases. Natural herbal plants have historically been utilized to treat a range of diseases, either directly or indirectly through their bioactive metabolites [2]. The unrivaled chemical variety of natural products, whether as pure chemicals or as standardized plant extracts, provides unlimited possibilities for new medicine development [3]. Researchers are increasingly turning to folk medicine for new ideas on how to develop stronger antibiotics for microbial illnesses [4]. The urge to evaluate the antibacterial activity of medicinal plants arose in response to the increasing failure of chemotherapeutics and antibiotic resistance exhibited by pathogenic microorganisms. Since the beginning of time, man has used a variety of plants and their diverse components to avoid and treat various illnesses [5,6].
According to a World Health Organization (WHO) research, medicinal plants are utilized for basic health care by 80% of the world's population [7]. Since ancient times, herbal plants have been used in various health remedies globally. Medicinal plants have a wide range of bioactive compounds that can be used to treat chronic and infectious diseases [8]. To assess the efficacy of medicinal plants, evidence from places where they are still used as medicine must be obtained. Alkaloids, flavonoids, tannins, and phenolic acids are the most important physiologically active compounds in medicinal plants, and they have physiological effects on the human body. These plants are well known as traditional medicines in rural areas of many poor countries [9]. Traditional therapists think that treatment derived from medicinal plants is more effective and less expensive than modern pharmaceuticals. In underdeveloped countries, low-income people, such as farmers and people from small villages, frequently use traditional medicine to treat common ailments [2,10,11].
Peganum harmala L belongs to family Zygophylaceae is a species of Peganum, also known as Alharmal, is one of the most valuable medicinal herbs [2,12]. Its many components are used in traditional medicine to treat a number of human illnesses, including lumbago, asthma, colic, jaundice, and as an emmenagogue stimulant [13]. It is a glabrous perennial herbaceous plant that thrives in semi-arid range terrain and sandy soils predominantly around the globe [4,6]. It can grow to a height of 0.8 meters. Alkaloids, flavonoids, and anthraquinones (antimalarial) are some of the well-known phytochemicals found in P. harmala [14,15]. It contains up to 4% total alkaloids [1]. P. harmala L has been demonstrated in several studies to have pharmacological and biological characteristics such as antibacterial, antifungal, and monoamine oxidases (MAO) [16].
P. harmala has anti-carcinogenic, vasodilator, anti-protozoal, and anti-inflammatory effects [1,17,18]. People in West Asia used to burn P. harmala seeds to produce smoke that protected them from voodoo [19]. Because of their analgesic and antibacterial properties, their fruits are used in traditional medicine. P. harmala has traditionally been used in traditional medicine to treat a wide range of conditions, including hepatitis B, asthma, lumbago, and colic [15]. The hallucinogenic -carboline alkaloids harmine, harmaline, harmalol, harmalacinine, isoharmin, and 8-glucosyloxyharmine are discovered in seeds [20]. Smoke is used as a disinfectant, an air cleaner, and a cold remedy. Research revealed the chemical composition and antibacterial activity of this smoke, however, the technique and temperature used to produce and analyze the smoke were still not clear [21].
However, little work has been done on phytochemical extracts as far as antimicrobial activities are concerned. Here we have focused on antimicrobial activities of different solvent extracts such as n-hexane, n-butanol, ethyl acetate, methanol, and water of P. harmala using stem and seeds by disc diffusion method. Ethyl acetate fraction was found more effective among the tested solvents and showed maximum activity against Staphylococcus aureus and Escherichia coli while n-butanol and water fractions gave maximum activity against S. aureus and Klebsiella pneumonia respectively. The water fraction has the greatest antifungal activity against Candida albicans. In the case of stem, ethyl acetate was more active against Bacillus subtilis and S. aureus while n-butanol was against K. pneumonia and E. coli. Methanol is more effective than ethanol against C. albicans. Seed extracts outperformed stem extracts in terms of efficacy. The extracted samples of ethyl acetate, butanol, and water have shown good antimicrobial activity against the tested bacteria, hence these solvents are recommended for future studies.

Plant materials collection
The present study was conducted at the Institute of Biotechnology and Genetic Engineering (IBGE), University of Agriculture, Peshawar Pakistan. P. harmala seeds were bought from a local market in Bannu, Khyber Pakhtunkhwa, Pakistan. 200 grams seeds were collected and then were grown in pots at the greenhouse of IBGE. 4 plants per pot are grown and a total of 22 pots were prepared. Stems were collected to obtain extracts from growing plants. All the plants were healthy and used for further experimental work. Plant materials were washed and dried at room temperature.

Crude extract preparation
The dried plant components, both seed and stem were ground into a fine powder using a grinder. The powdered components were soaked in methanol for one week. During this period, the plant components were combined daily to ensure that the bioactive compounds were fully dissolved. The plant materials were filtered and dried under reduced pressure in a rotary evaporator (Lab Tech EV311H). The entire process was repeated three times, each time combining all fractions. After that, the extract was transferred to a separating funnel and blended with 250-300ml of distilled water. An exact amount of n-hexane was added, shaken, and allowed to stand for 5-10 minutes. The organic layer was collected, and the process was performed three times more, each time with the addition of a fresh organic solvent. A variety of fractions were separated by adding n-butanol and ethyl acetate to water one at a time. All of the organic layers were then dried at reduced pressure using a rotary evaporator (Lab Tech EV311H) [22].

Fractionation
Eight to ten grams of crude methanolic extract from both seed and stem samples were kept for biological/pharmacological activities. The remaining was dissolved in 300 ml distilled water and was shifted to a separatory funnel and then added 300 ml of n-hexane. Shaking of the separatory funnel was done to separate the two layers of water and n-hexane. The n-hexane fraction formed the upper layer as it is lighter than water. The upper n-hexane layer was collected and the second layer of water was fractioned two times more with n-hexane. All the fractions of n-hexane were collective, filtered through Whatman filter paper, and dried through the rotary evaporator to obtain n-hexane fraction. The same fractionation procedure was conceded for ethyl acetate and n-butanol to obtain its extracts. The aqueous layer at the end was desiccated using a rotary evaporator. In this way, crude methanol; n-hexane, n-butanol, ethyl acetate, and aqueous fractions were obtained. A similar procedure was used for the stem parts also to get their extracts [22].

Media preparation for culturing
Nutrient agar media and nutrient broth were used during the study. Agar media was used for microbial culturing and growth while the nutrient broth was used for standardization and incubation (Tables 1 and 2). The required ingredients of the nutrient agar and nutrient broth were dissolved in distilled water and poured into conical flasks. All the media flasks and test tubes were sealed with cotton buds and then sterilized in an autoclave (Labtron) at 1.5 pounds pressure and 121˚C temperature for 15 min. After sterilization, the media was poured into Petri plates under aseptic conditions in the laminar flow chamber. The plates were then stored in an incubator (BIOBASE Biodustry Shandong, china) at 37˚C for 4 hours for later use. The nutrient broth in flasks (approximately 20 ml flask -1 ) was used for shaking incubation of microorganisms and nutrient broth in test tubes for standardization of microbial cultures [4,22].

Microorganisms
Antimicrobial activity of different solvent extracted samples from seeds and stem of P. harmala was tested against four bacterial and one fungal strain (Table 3). All microbial stock cultures were freshened by streaking using a sterile inoculation loop on nutrient agar medium plates in a laminar flow hood (BIOBASE), incubated at 37˚C for 24 hrs. After 24 hours, the streaked cultures were again sub-cultured on medium plates and incubated at 37˚C for 24 hrs. The second streaked cultures were inoculated into nutrient broth in flasks and subjected to shaking incubation for 18 hours at 37˚C (200 rpm). The selected bacterial and fungal strains for this study were B. subtilis, K. pneumonia, S. aureus, E. coli, and C. albicans (Table 3).

Antibacterial and antifungal assay
The antibacterial activity of different solvent extracted samples of P. harmala was carried by disc diffusion assay as described in Bauer et al. 1966 and antifungal activity by Ramdas et al. 1998 against different bacterial and fungal strains [23,24].

Disc diffusion susceptibility method
Nutrient agar media plates were seeded with 18-24 hours cultures of microbial inoculums (a standardized inoculum 1-2 × 107 CFU ml-1 0.5 McFarland Standard). Whatman No. 1 filter paper discs (6 mm in diameter) were placed with the help of sterile forceps on the media and then plant extracts in concentrations of 1 and 2 mg disc-1 in 6 and 12μL volume were applied on the discs. Antibiotics (6 μL disc-1) as positive control and DMSO (6 μL disc-1) as negative controls were also applied on the discs. Inoculated plates were then incubated at 37˚C for 18-24 hrs. The next day, zones of inhibition were recorded as an area in millimeters (mm) around the discs in each plate [4].

Positive controls
Erythromycin and ciprofloxacin were used as positive control for Gram-positive and Gramnegative bacteria. Clotrimazole was used as a positive control for fungal strain.

B. subtilis response towards different extracts
The study was undertaken to evaluate the antimicrobial activities of different solvent extracted samples from stem and seed of P. harmala against four bacterial species including both grampositive and gram-negative and one fungal species. N-hexane, ethyl acetate, n-butanol, methanol, and aqueous extracts from stem and seeds of P. harmala were tested against B. subtilis using disc diffusion assay (Fig 1). B. subtilus showed high susceptibility in the case of n-butanol and ethyl acetate extracted samples from stem extracted samples at lower and higher concentrations i.e. 1 and 2 mg discs -1 . N-butanol extracted samples revealed the highest inhibitory activity (42.97% ZI) at a concentration of 2 mg disc -1 . Ethyl-acetate extracted samples showed 42.10 percent inhibitory activity at 2 mg disc -1 . Methanol showed 26.31 percent inhibitory activity at higher concentration i-e 2 mg disc -1 , While 21.92% activity was shown at lower concentration i-e 1mg disc -1 . Aqueous and n-hexane extracted samples showed the lowest activities (17.21% and 24.55% ZI respectively) at higher concentrations (2 mg disc-1) while lower concentrations (0 and 18.42% ZI) were observed (Fig 1). Compared with stem-derived extracts nbutanol and ethyl acetate again showed the highest activity in the case of seed extracted samples against B. subtilus at lower and higher concentrations i.e. 1 and 2 mg discs -1 . N-butanol extracted samples revealed the highest inhibitory activity (57.00% ZI) at a concentration of 2 mg disc -1 . Ethyl-acetate extracted samples showed 52.63 percent inhibitory activity at 2 mg

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disc -1 . Methanol showed 57.89 percent inhibitory activity at higher concentration i-e 2 mg disc -1 and no activity was shown at lower concentration i-e 1mg disc -1 . N-hexane and aqueous extracted samples showed the lowest activity (28.05% ZI each) at lower concentrations (1 mg disc -1 ) and (34.21% and 40.34% ZI) at higher concentrations compared with other samples (Fig 1).

K. pneumoniae response towards different extracts
The same stem and seeds-derived extracts of P. harmala including n-hexane, ethyl acetate, nbutanol, methanol, and aqueous extracts were further tested against K. pneumoniae, using disc diffusion assay (Fig 2). It is clear from the data that K. pneumoniae was highly susceptible to nbutanol extracted sample both at 1 and 2 mg disc -1 concentration. The n-butanol fraction showed the highest inhibitory zone (24.55% ZI at 1 mg disc -1 and 32.44% ZI at 2 mg disc -1 ). Similar activity was observed in the case of n-hexane and aqueous extracted samples (19.28%, 24.55%, and 15.78%, 27.18%). Methanol and ethyl acetate extracted samples though reduced the microbial growth i-e 13.15% and 21.92% and that of ethyl acetate is 14.02% and 18.42% respectively (Fig 2). Compared with stem extracted samples, seeds extracted samples showed good results. K. pneumoniae was highly susceptible to aqueous extracted samples both at 1 and 2 mg disc -1 . The aqueous fraction showed the highest inhibitory zone (51.73% ZI at 1 mg disc -1 and 61.39% ZI at 2 mg disc -1 ). The n-butanol and methanol extracted samples revealed the same activity against K. pneumoniae (52.63% and 57.00% and 53.50% and 54.36% ZI for both n-butanol and methanol extracted sample) at 1 and 2 mg disc -1 respectively). N-hexane and ethyl acetate extracted samples though reduced the microbial growth i-e 35.94% and 38.07% and that of ethyl acetate is 40.34% and 52.63% respectively (Fig 2).

S. aureus response towards different extracts
The data regarding the antibacterial activity of n-hexane, ethyl-acetate, n-butanol, methanol, and aqueous extracted samples from the stem of P. harmala against S. aureus revealed that S. aureus is highly susceptible to all extracts of P. harmala. By increasing the concentration of different extracts, the degree of inhibition also increased. All the extracted samples were effective against S. aureus and inhibited the growth at both higher and lower concentrations (1 and 2 mg disc -1 ). Ethyl acetate extracted samples showed the highest inhibitory zones i.e. 46.66% at 2 mg disc -1 concentration. The lowest inhibitory zones were observed when n-butanol extracted samples were applied (22.20% at 2 mg disc -1 ) compared to a positive control (Fig 3). Comparing the antibacterial activity of n-hexane, ethyl-acetate, n-butanol, methanol and aqueous extracted samples from the seeds of P. harmala against S. aureus with stem extracted samples revealed that S. aureus is highly susceptible to all extracts of P. harmala. With increasing concentration of different extracts, an increased in the degree of inhibition was observed. Ethyl acetate extracted samples showed the highest inhibitory zones i.e. 81.10% at 2 mg disc -1 concentration. The lowest inhibitory zones were measured by n-hexane extracted samples (48.86% ZI at a concentration of 2 mg disc -1 ) when compared with positive controls (Fig 3).

E. coli response towards different extracts
The data regarding the antibacterial activity of n-hexane, ethyl-acetate, n-butanol, methanol, and aqueous extracted samples from the stem of P. harmala against E. coli by disc diffusion assay was also recorded (Fig 4). The data showed that all the solvent extracted samples gave significant activity. The n-butanol and methanol fraction reduced the growth of E.coli at both concentrations i-e 1mg and 2 mg disc -1 (27.93%, 37.61% and 23.64%, 35.48% respectively). Both n-hexane and aqueous extracted samples significantly reduced (33.32%) the growth of E.
coli at higher concentrations. Compared with stem extracted samples, seeds extracted samples showed good activity except for n-hexane which did not restrict the growth of E.coli. The ethyl acetate and aqueous fraction reduced the growth of E.coli at both concentrations i-e 1 mg and 2 mg disc -1 (46.22%, 32.25% and 61.29%, 41.93% respectively). The n-butanol extracted samples significantly reduced the growth of E. coli i-e 56.96% ZI at a concentration of 2 mg disc -1  and no activity were shown on lower concentration i-e 1 mg disc -1 , followed by crude methanolic extracted samples (53.74% ZI) at the highest concentration of 2 mg disc -1 and no activity were shown on lower concentration (Fig 4).

C. albicans response towards different extracts
The data regarding the antifungal activity of n-hexane, ethyl acetate, n-butanol, methanol, and water extracted samples from the stem of P. harmala against C. albicans by disc diffusion assay showed that all five samples can reduce the growth of C. albicans at 1 and 2 mg/disc concentrations ( Fig 5). Among these solvent extracted samples, methanol fraction showed the highest inhibitory activity (43.80%) at higher concentrations. Both n-butanol and n-hexane extracted samples reduced the growth of C. albicans (34.28% at the concentration of 2 mg per disc), while ethyl acetate and aqueous extracted samples showed the lowest inhibitory zones i.e 25.71% and 20.94% at higher concentration compared to positive control. Compared with stem extracted samples, in seeds extracted samples, the aqueous fraction showed the highest inhibitory zone i.e. 81.88% at 2 mg disc -1 concentration. Ethyl acetate and methanol extracted samples reduced the growth of C. albicans (78.08% and 74.28% at concentration of 2 mg per disc), while n-butanol and n-hexane extracted samples showed the lowest inhibitory zones i.e 59.02% and 40.00% at 2mg per disc as compared to a positive control (S1 Fig).

Discussion
Traditional medicines from various countries, which are the main source of complementary and alternative medicine, have recently shown promise in the treatment of a wide range of human diseases. The therapeutic approach of traditional medicine is heavily reliant on medicinal plants. P. harmala is a well-known medicinal plant with numerous pharmacological effects, including cardiovascular, neurologic, antimicrobial, anticancer, gastrointestinal, and antidiabetic properties. Several studies in modern phototherapy have confirmed some of the pharmacologic properties of this plant that have been documented in traditional Iranian medicine https://doi.org/10.1371/journal.pone.0265206.g005 [16]. According to El-Saad El Rifaei, the compounds extracted from P. harmala plant have a variety of medical properties, including the anti-inflammatory effects of pitolin [25]. He confirmed antibacterial/antifungal, antifever, and antiparasitic effects in P. harmala. The majority of studies on P. harmala medical effects have concentrated on parasite infections. Levchenko was among the first to report on the medicinal properties of P. harmala alkaloids in bovines [20]. In this study, the antibacterial and antifungal activities of different solvent extracts were measured for bacterial and fungal strains by the disc diffusion method.  [26]. They also used different solvent extracts against a wide-range of microorganisms. The same bacterial strains were reported by another group, and the antimicrobial activity was tested on 11 different bacteria using the agar diffusion method [27]. The water fraction had the highest activity against C. albicans (60.00 and 81.88%). In the case of the stem, Ethyl acetate was more effective against B. subtilus (38.57 and 42.10%) and S. aureus (36.66 and 46.66%) while n-butanol showed maximum activity against K. pneumonia (24.55 and 32.44%) and E. coli (27.93 and 37.61%). Methanol was found more effective against C. albicans (25.71 and 43.80%). Similar results were obtained by Hayet et al (2010) who also analyzed the antibacterial, antiviral and antioxidant activity of P. harmala against gram-positive and gram-negative bacteria [28]. These results are in contradiction with Dastagir and Hussain who have reported that the n-hexane extracts of Zygophyllaceae and Euphorbiaceae families showed the highest toxicity as compared to methanolic extracts [3]. Al-Izzy et al.
(2010) also reported that the aqueous fraction from P. harmala was effective in the inhibition of the growth of these microorganisms [29].

Conclusion
P. harmala, a versatile traditional medicinal plant with a diverse range of bioactive compounds, is an abundant source of bioactive compounds. Because little research has been conducted on the biological activity and potential medicinal applications of phytochemical compounds. More research could result in the development of new high-potency medications. This study demonstrates P. harmala potential for usage in innovative therapeutic drugs and establishes the framework for future medicinal plant research and antibacterial activity of P. harmala extracts against drug-resistant/clinically significant microbes.